The enzymatic conversion of sugar into acids with the help of carbohydrate oxidase and catalase finds many technological applications, particularly in the food industry. In some applications the carbohydrate oxidase is used to remove oxygen from a food product in order to preserve its quality. In other applications, the reduction of sugar content of the food product is desired.
The enzymatic conversion of sugar into acids involves an oxidation/reduction reaction, catalyzed by carbohydrate oxidase, in which oxygen serves as an electron acceptor. The oxygen is reduced to hydrogen peroxide (H2O2): sugar+O2+H2O→sugar acids+H2O2. The enzyme catalase catalyzes the reaction: H2O2→H2O+½O2.
If the production of a sufficient amount of acids is desired, the addition of catalase is necessary for the removal of hydrogen peroxide, which is an inhibitor of carbohydrate oxidase. It is also required that the reaction medium is continuously supplied with oxygen because the latter is consumed by the reaction. The amount of oxygen can be used to determine the optimal incubation time for the process.
A well studied carbohydrate oxidase is glucose oxidase (EC 1.1.3.4, GOX). Gluconic acid can be obtained by transforming glucose into gluconic acid using glucose oxidase. This occurs via the production of glucono-δ-lactone in an aqueous media when oxygen is available. Furthermore, H2O2 is produced from the reaction, which effectively inhibits GOX at already very low concentrations. On this account, it is common that GOX is used in combination together with the enzyme catalase (EC 1.11.1.6, CAT), which is capable of converting H2O2 into H2O and oxygen (Miron et al., 2004, Wong et al., 2008).Reaction of GOX: glucose+O2+H2O→gluconic acid+2H2O2 and of CAT: H2O2→H2O+½O2.
Due to the acid production, the enzymatic reaction process is generally limited by consecutive lowering of the pH-value, finally leading to inactivation of the enzyme, if no buffering substances are added (Miron et al, 2004). Thus, in biotechnological applications of enzymatic gluconic acid production by means of GOX and CAT, the pH is generally stabilized within the optimum range of enzyme activity by the addition of buffering substances or basic substances to achieve maximum transfer rates, as, for example, pointed out in WO-A-9635800 and DE-A-2214442.
In most ready-to-drink beverages (e.g. soft drinks, fermented drinks), the acid content as well as the sugar-to-acid ratio has to be in a defined, narrow range, to achieve an acceptable or even optimized sensorial impression. In the case of ready-to-drink beverages, the optimal sugar-to-acid ratio can be achieved by the production of sufficient amounts of acid under optimized reaction conditions by means of carbohydrate oxidase.
Although moderate glucose concentrations are applied in many GOX applications, highly concentrated glucose solutions are suitable as a substrate as well. In beverage concentrates, from which above mentioned ready to drink beverages can be obtained by dilution with water, the acid concentration (as well as the sugar content and all other ingredients) is several times higher compared to ready-to-drink beverages, leading to a much lower pH of the concentrate compared with the ready-to-drink beverage produced from it.
Under recommended optimal reaction conditions (recommended temperature and/or pH range), it is not possible to generate sufficient amounts of gluconic acid required for the beverage concentrate before the pH value is too low to obtain further enzymatic activity. Therefore, these applications resort to the addition of a buffering or basic substance to keep the pH of the concentrate constant and within the optimum enzyme activity range. In the case of beverages however, the use of buffers or bases to maintain the pH within the optimum range is not always suitable due to the possible negative sensorial impact.
For commercial GOX-preparations, recommended reaction conditions in terms of pH are in the range 4 to 7 pH, independent of the enzyme origin. Like any other enzyme, GOX from different origins can differ in their structure and hence their optimum conditions (Miron et al., 2004). GOX is mainly produced by Aspergillus or Penicillium subspecies. Almost all GOX preparations available on the market are produced by Aspergillus Niger (Handbook of Food Enzymology). For GOX from Aspergillus Niger, the pH of maximum stability was found to be around 5.5 (Miron et al., 2004). At pH lower than 3, the half-life of commercial Aspergillus Niger GOX has been found to be less than 20 minutes under assay conditions (Hatzinikolaou et al., 1996). The optimum temperature of GOX from various microbial sources has been reported to be between 25° C.-60° C. (Gibson et al., 1964, Wong et al., 2008, Bankar et al., 2009). Thus, it is usually the case that shifting the reaction conditions out of the optimum stops the reaction almost completely.
Several patents describe the combined use of GOX/CAT for the production of gluconic acid in beverages. For example, WO-A-2010106170 describes the use of GOX to produce an acidic beverage. The authors recommend reaction temperatures between 25° C. and 45° C. and the addition of a base to maintain the pH at a suitable constant value between 3.0 and 9.0 to increase the yield of gluconic acid. EP-A-0017708 suggests the use of reaction temperatures between 0° C. and 10° C. for the production of gluconic acid with immobilized GOX/CAT combination. The applicants emphasize that the pH value must remain constantly within the optimum region of pH 4-7, e.g. by means of the automatic addition of NaOH during the process. WO-A-97/24454 relates to the production of gluconic acid from glucose. The authors further recommend maintaining the pH of the glucose solution at from about 5 to about 7. WO-A-03/031635 describes the formation of calcium gluconate by converting glucose in gluconic acid in the presence of a calcium base, such as calcium oxide, calcium hydroxide and/or calcium carbonate, to neutralize the gluconic acid and to serve as calcium source. Thus, the processes claimed in these applications involve working under optimum enzyme activity conditions, achieved by buffering the pH to prevent inhibition due to low pH-values. WO-A-2009016049 describes a method for impeding oxidation reactions in food products by production of maltobionate from starch or maltose by an enzymatic process. Maltose is converted to maltobionate in the presence of carbohydrate oxidases, such as aldose oxidase, cellobiose oxidase, pyranose oxidase and hexose oxidase. Furthermore, catalase may be added to eliminate unwanted H2O2.
Accordingly, an object of this invention is to overcome the disadvantages outlined above and to provide a process of preparing a concentrated liquid foodstuff having a sufficient amount of acids without the addition of taste deteriorating buffering or basic substances, which control the pH during the sugar oxidation process.